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In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice
Published on: November 22, 2017
In situ architecture of developmentally programmed mitophagy reveals ER-phagophore membrane continuity
Yufei Huang1,2, Tianyu Zheng1,2, Xiaofeng Sun3
1Department of Chemical Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen, China.
None:
Selective mitochondrial clearance by autophagy (mitophagy) is essential for development and cellular homeostasis. However, how phagophores acquire sufficient membrane to engulf large mitochondria remains poorly understood. Here, we studied the in situ architecture of forming mitophagosomes in the developing Drosophila intestine by combining cryo-electron tomography (cryo-ET), serialized on-grid lift-in sectioning for tomography (SOLIST), cryo-focused ion beam (cryo-FIB) milling, and volume electron microscopy. Our data reveal that the endoplasmic reticulum (ER) forms continuous membrane connections with the phagophore during mitophagosome formation. In Vps13D mutant enterocytes, stalled mitochondrial phagophore membrane expansion is associated with an accumulation of persistent ER-phagophore membrane continuities. Together, our findings support a model in which the ER can establish direct membrane continuity with the phagophore to facilitate rapid mitophagosome formation.Abbreviation: APF: after puparium formation; BLTP: bridge-like lipid transfer protein; CCS: cleaning cross-section; cryo-ET: cryo-electron tomography; cryo-FIB: cryo-focused ion beam; CTF: contrast transfer function; ER: endoplasmic reticulum; HPF: high-pressure freezing; mitolysosome: autolysosome containing a mitochondrion; mitophagophore: mitochondrial phagophore; mitophagy: selective mitochondrial clearance by autophagy; NGS: normal goat serum; OMM: outer mitochondrial membrane; OsO4: osmium tetroxide; PB: phosphate buffer; PBSTx: PBS containing 0.3% (w:t) Triton X-100; RCS: regular cross-section; RT: room temperature; RT-FIB-SEM: room-temperature focused ion beam scanning electron microscopy; SOLIST: serialized on-grid lift-in sectioning for tomography; TLD: Through-the-Lens Detector.
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